Autosomal dominant polycystic kidney disease (ADPKD) is the most common lethal genetic disease inherited as a dominant trait in humans, with a prevalence of 1:1,000 live births. The disease afflicts approximately 6 million people world-wide, and accounts for 5–7% of all patients on dialysis in the United States (Gabow, 1984, Ann. Intern. Med. 101:238–247). Mutations in the PKD-1 gene account for 85% of ADPKD, while mutations in the PKD-2 gene account for 10% of the disease. In both cases, ADPKD is characterized by progressive, massive cystic enlargement of renal tubules resulting from increased proliferation, aberrant secretion, altered membrane protein polarity, and extracellular matrix abnormalities correlated with a failure to down-regulate certain fetal genes after birth (Wilson, 1996, In Polycystic Kidney Disease, Oxford Clinical Nephrology Series, Watson M. L. and Torres V. E. eds. p. 124–163).
Approximately 50% of patients who inherit a mutant PKD-1 gene will develop endstage renal failure, typically in the 5th decade of life, necessitating renal replacement therapy by dialysis or transplantation. Since progression is usually slow and is a consequence of gradual loss of renal function as cysts continue to enlarge and destroy intervening normal renal tubules, this presents a window of opportunity for potential drug therapies that would inhibit cyst expansion.
The PKD-1 gene maps to human chromosome 16p13.3, has 46 exons, encodes a 14.5 kb transcript with a 12,912 basepair open reading frame and translates into a 4303 amino acid (≧462 kDa) protein, referred to as “polycystin-1” (European PKD Consortium, 1994, Cell 77:882–894; International PKD Consortium, 1995, Cell 81:289–298). The predicted amino acid sequence of the expressed protein suggests that the first 23 amino acids at the N-terminus act as a signal sequence, followed by two cysteine-flanked leucine rich repeats (LRR) which are strongly predictive of an extracellular location, protein—protein interactions and adhesion properties (Hughes et al., 1995, Nat Genet 10:151–160). Other putative extracellular domains include a C-lectin-like motif and a region with high homology to the receptor for egg jelly of sea urchins (REJ), implying potential calcium influx regulation (Moy et al., 1996, J. Cell. Biol. 133:809–817). The protein has several regions of high hydrophobicity and predicts 9–11 transmembrane domains and an intracellular carboxy terminal tail of 226 amino acids with putative binding sites for signal transduction molecules, including a SH2 site for tyrosine phosphorylation (YEMV) and two putative protein kinase C sites (RSSR) for serine phosphorylation.
The polycystin-1 protein is localized to areas of contact between the cell and matrix shortly after adhesion to type I collagen matrix. In addition, there is co-localization with defining focal adhesion proteins, namely α2β1-integrin, vinculin, α-actin, talin, paxillin, focal adhesion kinase (pp125FAK) and pp60c-src (Wilson et al., 1998, J Cell. Biol. Vol. 9: 358A). Similar basally located polycystin-1-containing bodies have been demonstrated in vivo in human fetal ureteric bud epithelia in cell membrane regions in contact with type I collagen.
The overall predicted structure of the polycystin-1 protein and in vitro results with regard to matrix adhesion and phosphorylation assays, suggest that polycystin-1 functions as a matrix receptor mediating transfer of information from the extracellular matrix to the actin cytoskeleton, resulting in signal transduction that culminates in the nuclear regulation of gene transcription. This is suggested by the findings that all PKD-1 mutations mapped to date would predict the translation of a truncated protein product, lacking varying amounts of the C-terminal domain (CTD), including a potential SH2 site, and mutations that result in the failure to down regulate fetal expression of the β2 subunit of NaK-ATPase with consequent disruption of membrane polarization of NaK-ATPase (Peral et al., 1996, Am. J. Hum. Gent. 58:86–96). Of additional interest, ADPKD epithelia have lower levels of PKD-1 tyrosine phosphorylation and fail to recruit FAK to the basally located multi-protein bodies.
In normal mature kidneys, NaK-ATPase is comprised of α1β1 heterodimers located at the basolateral membranes of renal tubules and is associated with vectorial Na+ export into the basal cell space (blood side) driving ion gradients for fluid reabsorption. In normal fetal kidneys and also in ADPKD kidneys, the β2 subunit of NaK-ATPase heterodimerizes with α1 subunits which are “mis” targeted to apical plasma membranes, thus driving fluid secretion (Wilson et al., 1991, Am. J. Physiol. 260:F420F430). The failure to repress β2 transcription in adult kidneys results, therefore, in fluid secretion and expansion of renal tubule lumens into cysts.
Less is known about the PKD-2 gene, which has been mapped to human chromosome 4q 21–23, encodes a 5.4 kb transcript and translates into a predicted 110 kDa protein, “polycystin-2” (Mochizuki et al., 1996, Science 272:1339–1342). Unlike polycystin-1, the PKD-2 encoded protein has intracellular C— and NH— termini, 6 transmembrane domains with a putative EF hand and coiled-coil domain in the C-terminal region and putative SH3 sites in the N-terminal region. Yeast 2 hybrid studies have suggested potential interactions of PKD-1 and PKD-2 coiled-coil domains and some co-localizations have been suggested, however, in strict contrast to polycystin-1, polycystin-2 is not developmentally regulated (Tsoikas et al., 1997, Proc. Natl. Acad. Sci. 94:6965–6970; Qian et al., 1997, Nature Genet. 16:179–183).